I. Field of the Invention
The present invention relates to a system for discriminating a precipitation pattern of particles formed based on immunological agglutination and, more particularly, to a system for grouping or testing blood or discriminating an antibody and an antigen based on immunological agglutination.
II. Description of the Prior Art
A method of grouping blood from a precipitation pattern of blood corpuscles is already disclosed in Japanese Patent Publication No. 51-16798 (16798/1976). According to the blood grouping method, blood corpuscles to be inspected are separated by centrifugation. These blood corpuscles are recovered to prepare a 2 to 5% blood corpuscle suspension. This suspension and an antiserum are poured in appropriate quantities in a wine-cup-shaped reaction container. The container is then shaken, and allowed to stand. Thereafter, reaction mixture in the reaction container is subjected to centrifugation. The reaction containers are then relatively vigorously shaken, and subsequently relatively slowly shaken so that the reacted or agglutinated corpuscles are collected at the center region on the bottom of the containers. As a result, the precipitation pattern based on the agglutination of the blood corpuscles is formed on the bottom surface of the container. The precipitation pattern thus formed includes agglutination pattern and non-agglutination pattern. The agglutination pattern is formed as a result that the blood corpuscles are agglutinated with the antiserum and collected at the center region on the bottom surface of the reaction container. The non-agglutination pattern is formed as a result that agglutination does not occur, the blood corpuscles not agglutinated are dispersed in suspension. Accordingly, discrimination can be made on whether or not agglutination occurs by measuring the precipitation pattern optically.
The aforementioned blood grouping method is utilized exclusively for the ABO blood grouping since it includes the step of vigorously shaking the reaction container. The natural antibody used in this method agglutinates the blood corpuscles with large agglutinating force, and the agglutinated blood corpuscles are not separated from each other during such shaking step.
However, this method cannot be adopted for immunological method in which a reactant having a weak agglutinating force is involved a method of examining a variety of irregular antibodies, antigens or HBs antigens or the like. More specifically, if the agglutinating force is weak, when the reaction container is shaken, the blood corpuscles which have once agglutinated become separated from each other, and are not collected at the center of the reaction container.
Therefore, a method of employing a microplate with a number of small reaction containers or holes each having an inverted conical bottom surface is adopted for detection and grouping of the HBs antigens. This method, for example, detects and examines the HBs antigens with the steps described below, using, for example, a microplate having 10.times.12 pores.
(1) Droplets (each having a volume of 0.025 ml) of R-PHA buffer solution are added one by one to the respective holes of the microplate.
(2) A sample is diluted twice with a diluter, and ten types of samples having different dilution degrees are thus obtained. Two rows of such samples having different dilution degrees are prepared.
(3) One droplet of R-PHA buffer solution is added to the one row of the diluted samples, and one droplet (having a volume of 0.025 ml) of R-PHA inhibition solution is added to the other row of the diluted samples.
(4) After the solution in each hole is sufficiently shaked with a micromixer for 10 seconds, it is incubated at 37.degree. C. for 1 hour.
(5) One droplet (having a volume of 0.025 ml) of a 1% R-PHA cell floating liquid is added to each hole.
(6) The solution in each hole is sufficiently shaked with the micromixer for 10 seconds, and the R-PHA cell is uniformly suspended.
(7) After it is left to stand for 1 hour, avoiding shaking, at room temperature, the pattern formed on the bottom surface of each hole is examined.
According to this detecting method, since the microplate is sufficiently left to stand without shaking immediately before the detection, the agglutinated sample may not be separated, and the precipitation pattern based on immunological agglutination involving a reactant having relatively weak agglutinating tendency can be accurately formed on the bottom of each pore.
The inventor of the present invention has proposed as one of joint inventors in Japanese Patent Application No. 54-53370 a blood group detecting method which can sufficiently detect blood group based on immunological agglutinations involving natural antibody having strong agglutinating tendency and also can detect irregular antibody having weak agglutinating tendency. According to this blood group detecting method, blood corpuscles and reference antiserum reagent are contained in a reaction container having, for example, an inverted conical bottom surface, agitated, left to stand for a relatively short time (such as for approx. 30 minutes), and precipitation pattern is then examined to discriminate the blood group. According to this method, when the blood corpuscles to be examined react with the antiserum reagent, the precipitated blood corpuscles are thinly accumulated like snow on the inverted conical bottom as the blood corpuscles are agglutinated, and uniformly accumulated agglutination pattern is formed. When the blood corpuscles do not react with the antiserum reagent, the blood corpuscles do not agglutinate, but precipitate. When the blood corpuscles reach the inverted conical bottom, the blood corpuscles fall down along the oblique surface of the inverted conical bottom to collect at the center of the conical bottom surface, and non-agglutination pattern is formed. Accordingly, the blood group can be examined by photoelectrically detecting the difference of the precipitation pattern of the blood corpuscles formed depending upon whether or not the blood corpuscles react with the antiserum reagent.
However, the aforementioned various precipitation pattern discriminating methods fails to accurately detect the precipitating pattern formed on the bottom of the reaction container. According, for example, to the method disclosed in Japanese Patent Publication No. 51-16798 employing the wine-cup-shaped reaction container, the turbidity of the solution in the reaction container is measured by detecting the brightness of light passing through the reaction solution. That is, when the light beam passes through the solution, the degree of absorption of the light varies depending upon the amount of the blood corpuscles present in the path of the light beam, and the transmitted light beam is photoelectrically measured. In the embodiment shown in FIG. 33 of the Japanese Patent Publication No. 51-16798, light is incident from the top of the wine-cup-shaped reaction container. A mask having a center opening and an annular opening surrounding the center opening is disposed at the lower part of the reaction container, the light passed through the center opening is incident to the first photodetector, and the light passed through the annular opening is incident through a lens to the second photodetector. Accordingly, the brightness of light passed through the center of the reaction solution in the reaction container into the first photodetector represents the turbidity of the center region of the reaction solution, and the brightness of light passed through the periphery of the reaction solution incident to the second photodetector represents the turbidity of the periphery region of the reaction solution. Therefore, when the brightness of light passed through the center of the reaction solution is reduced than the reference value and the brightness of light passed through the periphery of the reaction solution is increased than the reference value, this is examined as "agglutination". When the brightness of light passed through the center and the periphery of the reaction solution does not change with respect to the reference value, it is examined as "non-agglutination". This precipitation pattern detecting method is considered to have no problem in case that the distance from the bottom surface of the reaction container to the center opening of the mask is shorter than the lateral expansion of the precipitation pattern. If the distance from the bottom surface to the center opening is longer than the lateral expansion, the light incident to the periphery of the reaction solution is scattered by the particles, tends to be incident through the periphery of the reaction solution to the first photodetector and the light incident to the center is scattered by the particles, and accordingly the brightness of light incident through the annular opening to the second photodetector is increased, with the result that an accurate light measurement cannot be conducted disadvantageously. More particularly, in case that it is impossible to dispose the opened mask sufficiently near at the bottom of the reaction container due to the system arrangement, or in case that the mask cannot be disposed sufficiently near at the reaction solution due to the necessity of irradiating the light from the bottom of the container and receiving the light from the top of the container, the light scattered with the particles in the reaction solution reduces the measuring accuracy to thus cause impossibility in the accurate examination. In order to eliminate such disadvantages, it is considered to increase the size of the reaction container so as to increase the difference of the turbidity of the solution. In this case, the quantity of sample is increased, and the sample cannot be substantially analyzed. An optical detecting system should be considerably complicated in order to obtain an accurate discrimination. Particularly when it is necessary to adopt a small reaction container so as to reduce the quantity of sample, it is difficult to decrease the size of the optical detecting system to thus cause a difficulty in the production and adjustment thereof.
Further, if the aforementioned turbidity measuring method is employed for the discriminating method disclosed in Japanese Patent Application No. 54-53370, the measuring accuracy cannot be raised. Particularly when the agglutination pattern and the non-agglutination pattern are automatically detected and discriminated, it needs a detecting equipment having considerably high accuracy. Moreover, the precipitation pattern formed on the bottom surface of the reaction container is not so always formed as to be distinct to be capable of distinguished from each other, and the intermediate state between the agglutination pattern and non-agglutination pattern may also be formed. It is necessary to examine the precipitation pattern including the intermediate pattern by accurately reading the precipitation pattern.